Separation of NSAIDs by Capillary Electrophoresis-Free-Samples

Question: Discuss about the Separation of NSAIDs by Capillary Electrophoresis. Answer: NSAIDs Non Steroidal Anti-inflammatory drugs or NSAIDs as the name suggests are drugs, which are non steroidal in nature. It helps to fight back against the inflammatory reactions and hence they are known as anti-inflammatory drugs. They act as an analgesic and helps to treat pain. It also has anti pyretic actions and suppress the signs and symptoms of inflammation. The classification of NSAIDs on the basis of their chemical nature (Conaghan 2012) Salicylates: Aspirin Sodium salicylate and diflunisal Propionic acid derivatives: ibuprofen, ketoprofen and naproxen Aryl acetic acid derivatives: diclofenac and ketorolac Indole derivatives: indomethacin and sulindac Alkanones: Nabumetone Oxicams: piroxicam and tenoxicam Anthranilic acid derivatives (fenamates): Mefenamic acid and flufenamic acid Pyrazolone derivatives: phenylbutazone, oxyphenbutazone and azapropazone (apazone) dipyrone (novalgine) Aniline derivatives (analgesic only): paracetamol Properties of Salicylates Aspirin is the most important example of Salicylates. It is gets readily absorbed from the stomach and during its passage from the upper part of the small intestine. Upon absorption, it gets distributed all over the body via diffusion in the body fluid, blood. Nearly 50?80% of the salicylate moves via binding with the plasma protein (albumin). Salicylate an active metabolite at times remains conjugated with glucuronic acid and glycine and gets excreted through the kidney and this excretion is further increased via the alkalinization of the urine (Rainsford 2013). Aspirin Mechanism of Action A family of very potent biological signaling molecule, Eicosanoids, acts as short-range messengers. It causes the localized action that is, it affects tissues near the cells that produce them. In response to the hormonal stimulation or other stimuli, Phospholipase A2 (an outer membrane protein of mammalian cell) cleaves membrane glycerophospholipids, releasing Arachidonate from the middle carbon of glycerol. Following this reaction, the enzymes present in the smooth endoplasmic reticulum (ER) converts arachidonate into prostaglandins (prostaglandin H2 or PGH2). The peroxides then convert PH2 into the other precursors of prostaglandins and thromboxanes (Lehninger 2012). A bifunctional enzyme, cyclooxygenase (COX) (also known as prostaglandin H2 synthase) promotes the conversion of the arachidonate into PGH2. Prostaglandin is a secondary mediator of type I hypersensitivity reactions. It is produced after the mast cell degranulation and thus causes more pronounced and long lasting effect. Prostaglandin D2 causes broncho constriction. The other prostaglandins caused vasodilatation, contraction of the pulmonary smooth muscles and platelet aggregation (Owen, Punt and Stranford 2013). In order to treat such inflammatory reactions caused by the secondary mediators, Aspirin is prescribed. Aspirin (acetylsalicylate) irreversibly inactivates the activity of the COX. It acetylates the Ser residue present at the active site of the enzyme and thus blocking the enzymes activity and this in turn inhibits the synthesis of prostaglandins and thromboxanes (Dovizio et al. 2013). NSAIDs like indomethacin and ibuprofen, inhibits the action of cyclooxygenases via the mechanism of competitive inhibition. They compete with the arachidonate with the active substrate binding site of COX and thus inhibiting the action of the enzyme on Arachidonate. Figure 1: Conversion of arachidonate into prostaglandins and thromboxane (Source: Lehninger 2012; pg 801) Apart from blocking the synthesis of the prostaglansinds, Aspirin also has other notable anti-inflammatory functions (Price et al. 2012; Imazio and Adler 2013). It blocks the action of kinins, which are mediated through the synthesis of prostaglandin Inhibits the adherence of granulocyte to the damaged vasculature and thus inhibiting the mast cell degranulation followed by redness and swelling It stabilizes lysosomal membrane and hence preventing the released of the lysosomal enzymes which has a proteolytic property Inhibits the migration of Poly Morpho Nuclear Neutrophil (PMN), leukocytes and macrophages at the site of inflammation Caffeine, an important NSAID Caffeine plays an important role in pain-related predicaments. It is by far the most widely consumed psychoactive (affecting the mind/behavior) substance round the world. The main action of Caffeine is attributed to its affect on the central nervous system (CNS). It has several stimulating effects on CNS like increased in the level of alertness and decreased in fatigue. It is also used to treat the or nullify the over-the-counter effects of the analgesics (pain-relieving medications). The major source of caffeine is tea, coffee, soda and chocolate. However, the existence of caffeine is also indicated in the drugs, which are used as a stimulant. Caffeine works via binding and blocking the adenosine receptors. Adenosine binds to adenosine receptors and normally induces sleep. Caffeine is known to have similar structural symmetry with that of the adenosine. It competitively inhibits the binding of the adenosine with receptors and on the other hand, it stimulates the release of the adren aline and increases the rate of heartbeat. This increase in the heartbeat and increase in the level of adrenaline reduce sleep and help to stay reenergized or active. Caffeine also reduces pain. Presence of caffeine has been found active in certain analgesic. The prominent presence of caffeine aggravates the pain-relieving effect of those drugs. In lower doses, dietary caffeine is used to prevent certain medications. It also interferes with the effectiveness of the acupuncture via manipulating the effect of adenosine (Syrovaya and Grabovetskaya 2015). Caffeine has been reportedly added into aspirin, acetominophen and other non-steroidal anti-inflammatory drugs (NSAIDs). It acts as an adjuvant to the analgesic effect and assists in the overall pain-relieving process of the main active drug. Most recent literature suggests that caffeine when used with NSAIDs, may provide fast relief from headache pain, but the same theory does not hold true for the postsurgical pain. Therefore, the use of analgesics with caffeine must be done with caution and under the supervision of medical practitioner. Caffeine amplifies the effects of NSAIDs via accelerating the normal chemical action of NSAIDs (Di et al. 2013). However, for some chronic caffeine users, sudden cessation of the use of caffeine can lead to the generation of withdrawal syndrome and thus accelerating the chronicity of headache and fatigue. On the other hand, several studies have shown that the people who are about to enter surgery, stopping the intake of caffeine for an extended spa n of time, and then again restoring its use on the day of surgery has led to occurrence of post headaches trauma (Mller et al. 2012). Still then, reduced caffeine intake is always recommended in order to reduced the chances of habit formation (Lpez-Cruz et al. 2013). Electrophoresis Electrophoresis is a process of protein separation based on the migration of the charged proteins in an electric field. In this analytical method of protein identification, proteins can be easily separated as well as visualized and thus enabling the researcher to ascertain the number of different proteins present in the mixture or purity of a particular protein mixture. The process also helps in the elucidation of several crucial protein properties such as its isoelectric point and approximate molecular weight. The process electrophoresis is carried out in gels that is made up of the cross-linked polymer, polyacrylamide. This molecular sieve, polyacrylamide gel, slows the migration of proteins based on their charge-to-mass ratio and shape (Lehninger 2012). Figure 2: Electrophoresis Gel Plate (Source: Lehninger 2012) Separation of NSAIDs by Capillary Electrophoresis Capillary electrophoresis (CE) is used for analytical method of protein separation or estimation. The majority of CE separations have been performed in the aqueous media. The aqueous medium is prepared with the help of the organic solvents due to their micellar effect in electrokinetic chromatography and capillary electrochromatography. Such effect improves the separation of hydrophobic compounds (Bonvin, Schappler and Rudaz 2014). The organic solvents as the separation media in CE are selected on the basis of several factors: Dielectric constant Viscosity UV-absorbance Capability to increase hydrophobic interactions However, non-aqueous electrolytes are also used as a medium and better solubility of hydrophobic analytes. Non-aqueous media gained importance in the field of identifying those compounds, which are insoluble in water or has poor water solubility, It has also been reported to improve the selectivity of the separation of compounds that have similar electrophoretic mobilities in aqueous media. Another interesting aspect of nonaqueous electrolytes is, such media has complete evaporability and thus, highly suitable for coupling CE with mass spectrometry. CE conducted with aqueous buffers contain high concentration of additives like cyclodextrins, cellulose derivatives and hence, not suitable for the mass spectrometric analysis. Non aqueous electrolytes can be easily prepared with volatile electrolytes (ammonium acetate, ammonia and acetic acid). Such volatile electrolytes has high end applications in mass spectrometry (Zhang et al. 2014). Chemicals and reagents used in CE of NSAIDs Methanol of HPLC grade (maximum 0.02% of water), Ammonium acetate of analytical grade, Ammonia, Acetic acid, Benzylic alcohol, Water (Milli-Q quality) (Millipore, Bedford, MA, USA), Piroxicam, Indomethacine, niflumic acid, ketoprofen, suprofen, sulindac, carprofen, indoprofen, flurbiprofen and naproxen are must for CE of NSAIDs. Other important chemicals are alclofenac, and tiaprofenic acid. All the solutions, which are used in CE of NSAIDS must be filtered using polytetrafluoroethylene (PTFE) membranes of 0.22 mm (Altria 2013). The process of CE of NSAIDs The electrophoretic separations must be carried out with the help of uncoated fused-silica capillaries with 50 mm internal diameter and 44 cm length. Before injection, the capillary used must be treated with alkaline solutions (1 M NaOH, 0.1 M NaOH), followed by water and then running buffer. For optimal separation, the CE buffer for NSAIDs must consist 50 mM ammonium acetate 13.75 mM ammonia in different organic solvents, preferably methanol. The applied voltage of the electrophoretic separation must range in between 25 kV. Such voltage range (+/- 25kV) is compatible with both methanolic buffer and other aqueous buffers. The subsequent UV detection after the separation must be performed at 280 nm (this is the standard range for protein). The injections of the NSAIDs must be done in a special hydrodynamic mode for a period of 2 seconds and corresponding amount inserted in each well must not be more than 5.3 nL. Prior the use, the capillary must be thermo stated at 25 degree Centigra de (unless otherwise stated). The test mixtures of NSAIDs must be prepared with extra caution via reconstituting in methanol. While the protein is being loaded into the well, the order must be noted simultaneously in a copy for proper estimation after the run. The electro osmotic flow must always be measured with a neutral marker or ladder, which must be inserted on one side of the well at the extreme right hand or left hand side of the well. The resolution (Rs) and plate number (N) must be calculated according to the standard expressions based on the peak width at half height (Galievsky, Stasheuski and Krylov 2014). The electro osmotic mobility of the methanol medium is lower than that of the effective mobilities of NSAIDs. Therefore, in methanolic system, the polarity of the electrical field needs to be reversed and the NSAIDs must be detected at the anodic end of the capillary and the electroosmotic flow then must moves in the opposite direction. For a standard test, a non-aqueous electrolyte that is made of 50 mM ammonium acetate and 13.75 mM ammonia mixed in methanol is best suited to resolve a mixture of NSAIDs at 25 degree C. The non-aqueous solution increases the separation selectivity of the NSAIDS in CE. If the proteins are more hydrophobic in nature then the selectivity of the separation is increased further. However, when more complex mixture is used, the temperature of separation must be higher that is at least 36o C. Moreover, the methanolic buffer must be mixed with 30% acetonitrile in order to get satisfactory separation for complex NSAIDs mixture at 35o C. Non-aqueous solution also provides better results in comparison to the aqueous solution when planning to separate protein mixtures of similar charge densities (Ali, Sanagi and Aboul?Enein 2014). Comparative Study between Capillary Electrophoresis and HPLC CE has similar mechanism of protein separation like normal electrophoresis but the only difference is, it is carried out inside a capillary. The small diameter of the capillary and its large aspect ratio (length/width) contribute more refinement in the separation. The capillaries were introduced in the process of electrophoresis as an anti-convective heat controlling innovation. The narrow capillary reduces the lateral diffusion and subsequently ensures that the temperature difference between the center of the capillary and the wall is same. Such negligible variance of temperature helps in proper separation and migration of the protein bodies under the action of the charged electronic field. Chromatography is defined as a process of separation of different components in the mixtures or solution based on the relative amounts of each solute distributed over the moving fluid stream known as mobile phase and a contiguous stationary phase. Here the mobile phase used is either liquid or gas while on the other hand, the stationary phase is solid or liquid (Smith 2013). High Performance Liquid Chromatography (HPLC) is a modern refinement in the chromatographic method. It is done with the help of a column of smaller diameter (Snyder, Kirkland and Glajch 2012). Within this column, the solvents are forced to move under high pressure. The high-pressure pumps speed up the movement of the protein molecules down the column based on their size and shape. By reducing the transit time on the column, HPLC limits the diffusional spreading of protein bands and thus improve the resolution. Incompressible silica or alumina microbeds are used as stationary phase in HPLC. The incompressible matr ix permits enhanced resolution and high flow rate. HPLC is used to resolve complex mixtures of both lipips and proteins whose properties differ slightly (Rodwell et al. 2015). Other different chromatographic methods include Partition Chromatography Size Exclusion Chromatography Absorption Chromatography Ion Exchange Chromatography Hydrophobic Interaction Chromatography Affinity Chromatography Both electrophoresis and chromatography are separation techniques. However, there lies certain difference between chromatography and electrophoresis. Electrophoresis consists of a stationary phase and a wet mobile phase. Here the stationary phase is a solid phase, and the wet mobile phase is either acidic or basic solution. Chromatography also has stationary phase and a mobile phase but in this case, the mobile phase is a liquid orgas phase. Chromatography is used in lipid separation and Electrophoresis is used in the separation of protein or DNA on the basis of their charge and molecular weight. Development of separation technique of NSAIDs The separation of the NSAIDS with the help of CE or other electrophoretic and chromatographic techniques is important because it helps in the size and charge based identification of the proteins. There are several NSAIDS which have very similar physical properties like sulindac , niflumic acid, piroxicam, flufenamic acid, alclofenac, flurbiprofen, tiaprofenic acid, suprofen, naproxen, ketoprofen, indomethacin, carprofen and indoprofen. The proper electrophoretic separation and subsequent identification of these proteins on the basis of the charge and mass will help the proper characterization of the chemical nature of the protein. Such characterization will be extremely beneficial in the end because it will help to determine the toxicity, side-effects and dosage (Sultana et al. 2012). Comparative study on the separation technique CE is the best possible way for the separation of the NSAIDS on the basis of their charge my mass ration. The use of the non-aqueous solution as a separation medium has further increased the rate of specificity of the separation. The use of CE over normal electrophoresis is more specific because in CE, the heat distribution is even and thus, promoting the proper and uniform migration of the protein. HPLC however, can also be used for the separation of the NSAIDS the liquid, which is used as the medium of separation in this technique may generate false positive results. Importance of the study The importance of the study of separation of the NSAIDS via CE lies in the fact, it has opened the new gateway towards the elucidation of the mass, charge and size of the proteins. Thus, characterization will subsequently help in proper use of these proteins in generation of anti-inflammatory drugs. Anti-inflammatory drugs are used to treat several inflammatory diseases. Inflammatory diseases if not treated on an urgent basis may become life threatening. On the other hand, several steroidal drugs, which are available in the market for the treatment of the inflammation, have deadly side effects (Fan and Morand 2012). Few of the common side-effects which are common with the prolong use of the steroidal drugs are Acne Blurred vision Cataracts or glaucoma Susceptibility towards diabetes Easy bruising Insomnia or difficulty in sleeping High blood pressure Cardiac problems Gain or loss in weight Increase appetite Increase growth of body hair (Ciriaco et al., 2013; Waldron et al. 2012) In case of the NSAIDs the side are present but are not as severe as those of the steroidal drugs. Moreover, the prolong use of such drugs do not lead to the habit formation. Due to the their low severity in the side-effects. NSAIDS are considered as the best possible agents for the treatment of the sudden inflammatory reaction. But, in order to formulate the medicines, the nature of the drugs or the characterization of the proteins are important. This characterization of the proteins is done with the help of the electrophoresis or capillary electrophoresis to be more precise. Such characterization of proteins helps in the proper identification and each and every NSAIDs which pose similar physical properties including same solubility, viscosity, more of action, colour etc. After the proper characterization pnly, these drugs can be used in the pharmacological industries for the proper formulation of the medicines. Moreover, such characterization may also be proved helpful in estimating the side-effects of the NSAIDs, their respective dose, the critical difference between the closely linked drugs and their rate of action the time of recovery (Lichtenberger et al. 2012). 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